Role of the Cytoplasmic Membrane in the Synthesis of Ribonucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Disrupted spheroplast preparations of Pseudomonas schuylkilliensis strain P contained fragments of cytoplasmic membrane and approximately 82% of the total cellular phospholipid. The protoplast-bursting factor (PB-factor), partially purified from pig pancreas, and a heat-treated pancreatic lipase fraction both inhibited ribonucleic acid (RNA) synthesis by disrupted spheroplasts but did not inhibit or only slightly inhibited RNA synthesis by intact cells or intact spheroplasts. The PB-factor preparation and the heat-treated pancreatic lipase fraction catalyzed partial (15 to 50%) deacylation of diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine in disrupted spheroplasts but not in intact spheroplasts. Phospholipase A activity was demonstrated in the PB-factor preparation by use of isolated phospholipids as substrates. Treatment of disrupted spheroplasts with the PB-factor preparation caused a 70% inhibition in oxidative phosphorylation and RNA synthesis, but had little effect on electron transport. Addition of adenosine-5'-triphosphate or adenosine-5'-diphosphate and a mixture of ribonucleosides after treatment with the PB-factor preparation partially restored oxidative phosphorylation but did not relieve the inhibition in RNA synthesis. The most reasonable explanation for the latter observation appears to be that the concentrations of newly synthesized nucleotides retained by the preparations with partially deacylated membrane phospholipids were insufficient to permit the synthesis of RNA.     

Dissolved organic carbon and fluorescence in Lake Hovsgol: factors reducing humic content of the lake water

 Lake Hovsgol is a large tectonic lake located in northern Mongolia, which has extremely transparent lake water. In our survey, the dissolved organic carbon of the lake water was 80–100 μM-C, and the fluorescence intensity in an excitation and emission matrix was very low. The brown color and high content of humic substances in river water flowing from a watershed consisting of grassland and forests rapidly declined in the coastal area of the lake. The decrease in humic content may be due not only to dilution by the lake water but also to flocculation and photobleaching. Among tectonic lakes in Asia, Lake Hovsgol would appear to have unique biological and hydrological features that reduce humic content and help to maintain water transparency. Received: June 25, 2002 / Accepted: January 10, 2003 Acknowledgments The authors acknowledge helpful discussion with Dr. J. Urabe. We thank Dr. T. Galbaatar, Mongolian Academy of Science, Mongolia, for his arrangements on the expeditions in 1999. We are also indebted to Mr. D. Hadbaatar, B. Ganbat, and the cruise staff of the R/V Suchbaatar for their assistance in the course of the study. This study was supported by Grant-in-Aid No. 09041159 and 13575034 for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Correspondence to:K. Hayakawa     

1H, 13C,and 15N resonance assignments of mouse lipocalin-type prostaglandin D synthase/substrate analog complex

Lipocalin-type Prostaglandin D synthase (L-PGDS) acts as the PGD₂-synthesizing enzyme in the brain of various mammalian species. It belongs to the lipocalin superfamily and is the first member of this family to be recognized as an enzyme. Although the solution and crystal structure of L-PGDS has been determined to understand the molecular mechanism of catalytic reaction, the structural analysis of L-PGDS in complex with its substrate remains to be performed. Here, we present the nearly complete assignment of the backbone and side chain resonances of L-PGDS/substrate analog (U-46619) complex. This study lays the essential basis for further understanding the substrate recognition mechanism of L-PGDS.     

Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633.

An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.     

Mechanism of foetal growth retardation caused by smoking during pregnancy

In order to clarify the mechanism of retarded foetal growth in smoking pregnant women, foeto-placental function and maternal nutritional condition were assessed. Dehydroepiandrosterone sulfate (DHAS) loading test, measurement of cotinine which is a major metabolite of nicotine and pathohistological examination of placental villi were also made to know the effect of smoking on utero-placental circulation. In heavy smokers, urinary oestriol and serum hPL levels were lower than those in non-smokers while the maternal nutritional condition was not different from that in non-smokers. In the DHAS loading test, heavy smokers showed lower conversion of DHAS to oestradiol. In the non-stress test (NST), bradycardia and/or loss of variability of baseline foetal heart rate were noted after smoking. Levels of cotinine in maternal blood and umbilical cord blood in heavy smokers were markedly higher than those in non-smokers. Microscopic examination showed atrophic and hypovascular changes of placental villi obtained from smoking mothers. These results suggest that the retarded fetal growth in heavy smokers is due to the impairment of utero-placental circulation as a result of the vasoconstricting effect of nicotine.     

Pleiotropic phenomena in autolytic enzyme(s) content, flagellation, and simultaneous hyperproduction of extracellular alpha-amylase and protease in a Bacillus subtilis mutant.

A mutant of Bacillus subtilis 6160 that had been isolated by its hyperproduction of alpha-amylase and protease lacked flagella and motility, and its content of autolytic enzyme(s) was reduced to one-third to one-fourth that of the parent. These phenotypic differences were completely co-transferred by the deoxyribonucleic acid (DNA) of the mutant when five DNA recipient strains of B. subtilis were transformed. The revertants, isolated by motility with a frequency of approximately 10(-7), recovered a normal level of autolytic activity and showed reduced productivity of alpha-amylase and protease. This point mutation allowed normal flagellin synthesis, spore formation, and rate of growth. The comparison of cell envelope of the mutant with that of the parent indicated that there was no significant difference except loss of flagella. Therefore the association at the cell surface of a group of extracellular proteins consisting of alpha-amylase, proteases, flagellin, and autolytic enzymes(s) seem to be coordinately regulated by the gene or seem to be affected coordinately by certain undetected alterations of the cell envelope.     

Transformation of Bacillus subtilis in α-Amylase Productivity by Deoxyribonucleic Acid from B. subtilis var. amylosacchariticus

Deoxyribonucleic acid (DNA) of Bacillus subtilis var. amylosacchariticus showed almost the same ability as B. subtilis Marburg to induce transfer of several genetic markers in DNA-mediated transformation. DNA-DNA hybridization data also showed an intimate relationship between the two strains. Genetic elements involved in the production of extracellular alpha-amylase (EC 3.2.1.1.) in B. subtilis var. amylosacchariticus were studied by using DNA-mediated transformation. Two Marburg derivatives, NA20(amyR2) and NA20-22(amyR1), produced about 50 and 10 U of alpha-amylase per mg of cells, respectively, whereas B. subtilis var. amylosacchariticus produced as much as 150 U of the enzyme per mg of cells. When B. subtilis var. amylosacchariticus was crossed with strain NA20-22 as recipient, transformants that acquired high alpha-amylase productivity (about 50 U/mg of cells) were obtained. Genetic analysis revealed that a regulator gene (amyR) for alpha-amylase synthesis was found in B. subtilis var. amylosacchariticus, as in the case of B. natto 1212 (amyR2) and B. subtilis Marburg (amyR1). The allele was designated amyR3; it is phenotypically indistinguishable from amyR2, but is readily distinguishable from amyR1. The presence of amyR3 was not sufficient for an organism to render production of an exceptional amount of alpha-amylase. Extra-high alpha-amylase producers could be obtained by crossing B. subtilis var. amylosacchariticus as donor with strain NA20 as recipient. The transformants produced the same or even greater amounts of the enzyme than the donor strain. Results suggest the presence of another gene that is involved in the production of the exceptional amount of alpha-amylase.     

Genetic Control of the Rate of α-Amylase Synthesis in Bacillus subtilis

The level of extracellular alpha-amylase (EC 3.2.1.1) of Bacillus subtilis Marburg was increased about fivefold by introducing the amyR marker from B. natto 1212 through transformation. amyR2 of B. natto 1212 has been assumed to determine a high level of alpha-amylase of the organism. The gene acts specifically on alpha-amylase synthesis but not on the production of other extracellular enzymes. alpha-Amylase of an amyR2-carrying strain was found to be quite similar to that of an isogenic amyR1-carrying strain in the thermostability and electrophoretic behavior of whichever amylase the strain produces. Marburg-type alpha-amylase (amyEm) or B. natto-alpha-amylase (amyEn). Anti-amylase serum titration indicates that a high level of the enzyme activity in the amyR2-carrying strain is caused by the existence of more enzyme rather than the presence of an enzyme having higher efficiency. This is supported further by the fact that amyR controls the synthesis of the amyE gene product in mutant M9, which synthesizes a temperature-sensitive-alpha-amylase, and in mutant M07, which secretes cross-reacting material. The results indicate that amyR regulates the rate of alpha-amylase synthesis.